Glycoproteins are important biomacromolecules that are biosynthesized through posttranslational glycosylation of newly fashioned proteins emerging from the ribosome. Interest in glycoproteins arises at many levels. A long-term goal of the growing field of chemistry-based glycobiology is the delineation of the consequences of glycosylation on critical properties such as protein folding, proteolytic stability, and cell adhesion (Imperiali, B.; O'Connor, S. E.; Hendrickson, T.; Kellenberger, C. Pure Appl. Chem. 1999, 71, 777-787; Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1-27; Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376; Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-2364; each of which is incorporated herein by reference). Such insights could explain why nature bothers to glycosylate otherwise functional proteins. Moreover, glycoproteins have potentially important clinical roles in the context of vaccines, diagnostics, and therapeutics. Indeed, erythropoietin, albeit a heterogeneous glycoprotein (Rush, R. S.; Derby, P. L.; Smith, D. M.; Merry, C.; Rogers, G.; Rohde, M. F.; Katta, V. Anal. Chem. 1995, 67, 1442-1452), is clinically valuable as a treatment for anemia, among other indications (Ridley, D. M.; Dawkins, F.; Perlin, E. J. Natl. Med. Assoc. 1994, 86, 129-135).
Many naturally occurring, medicinally important glycoproteins (cf., for example, erythropoietin [Ridley, D. M.; Dawkins, F.; Perlin, E. J. Natl. Med. Assoc. 1994, 86, 129-135] and gp120 [Geyer, H.; Holschbach, C.; Hunsmann, G.; Schneider, J. J. Biol. Chem. 1988, 263, 11760-11767]) display multiple glycosylation sites containing large oligosaccharide domains. However, given the complexity and variability of biological glycosylation pathways (Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-664; Roth, J. Chem. Rev. 2002, 102, 285-303; each of which is incorporated herein by reference), the isolation of homogeneous glycoproteins from natural sources in significant quantity is extremely difficult.
Numerous methods exist for the production of glycopeptides by chemical synthesis. For example, glycans have been introduced into peptides via amino acid “cassettes” with pendant protected saccharides (Chen et al., J. Am. Chem. Soc. 1998, 120, 7760-7769; Bezay et al., Angew. Chem. Int. Ed. 2001, 40, 2292-2295; van Ameijde et al., J. Chem. Soc.-Perkin Trans. 1 2002, 1042-1049; Ciommer et al., Synlett 1991, 593-595; Chiesa et al., Eur. J. Org. Chem. 2000, 3541-3554; Meinjohanns et al., Tetrahedron Lett. 1995, 36, 9205-9208; each of which is incorporated herein by reference), through enzymatic manipulations of glycopeptides (Unverzagt, Tetrahedron Lett. 1997, 38, 5627-5630; Witte et al., J. Am. Chem. Soc. 1997, 119, 2114-2118; Wang et al., J. Am. Chem. Soc. 1997, 119, 11137-11146; Arsequell et al., Tetrahedron: Asymmetry 1999, 10, 3045-3094; Mizuno et al., J. Am. Chem. Soc. 1999, 121, 284-290; Koeller et al., J. Am. Chem. Soc. 2000, 122, 4241-4242; Blixt et al., J. Am. Chem. Soc. 2002, 124, 5739-5746; each of which is incorporated herein by reference), or by conjugation of fully elaborated, complex saccharides to short synthetic peptides (Anisfeld et al., J. Org. Chem. 1990, 55, 5560-5562; Cohen-Anisfeld et al., J. Am. Chem. Soc. 1993, 115, 10531-10537; Meinjohanns et al., J. Chem. Soc.-Perkin Trans. 1 1998, 549-560; each of which is incorporated herein by reference). Larger O-linked glycopeptides have been synthesized using ligation techniques (Dawson et al., Science 1994, 266, 776-779; Liu et al., Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6584-6588; each of which is incorporated herein by reference) such as expressed protein ligation (Muir et al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-6710; Macmillan et al., Tetrahedron 2000, 56, 9515-9525; Tolbert et al., J. Am. Chem. Soc. 2000, 122, 5421-5428; each of which is incorporated herein by reference). Bertozzi and coworkers extended the scope of the “cassette” approach by applying native chemical ligation to the synthesis of a biologically active glycoprotein with two single-residue O-linked glycans (Shin et al., J. Am. Chem. Soc. 1999, 121, 11684-11689; incorporated herein by reference). Tolbert and Wong described the ligation of a 392-residue intein-generated peptide thioester and a dipeptide functionalized with a single N-acetylglucosamine residue. However, none of these approaches has allowed the assembly of complex glypeptides or glycoproteins multiply functionalized (e.g., multiply glycosylated) at designated sites.
Accordingly, there remains a need for novel synthetic methods for the preparation of homogeneous glycosylated, or otherwise post-translationally modified, peptides and proteins. Specifically, convergent, stereoselective, versatile methods for preparing such glycopeptides and/or glycoproteins are needed.